Post-translational fatty acylation of internal protein residues is a powerful but poorly understood biological mechanism for altering a protein's behavior. It is important in diverse physiologic strategies, for example, signal transduction The activation of Escherichia coli hemolysin (HlyA) toxin is used in this proposal as a model to study protein internal acylation. Pathogenic E.coli produce nontoxic proHlyA which is made toxic by the post-translational addition of 2 fatty acyl groups to internal residues, causing an extraordinary change in protein behavior. Acyl-acyl carrier protein (ACP) is the obligatory acyl-donor. HlyC is the internal protein acyltransferase, which forms an acyl-HlyC intermediate, evidence suggests an acyl-histidyl HlyC. Using a precise and direct assay, the reaction employing separately subcloned, purified proteins is a unique opportunity to explain the biochemical mechanism of an internal protein acyltransfer (the only one to be so studied) and its role in changing protein behavior. The following specific aims will be pursued with the goals of further understanding the biochemistry of protein internal residue acylation and associated changes in protein behavior and what defines an internal protein acylation site: 1. Site- directed mutation analysis, site-directed fluorescence, and chemical modification will be used to define the roles of several residues shown to be important in HlyC's catalytic function, and the acyl-HlyC intermediate will be characterized. 2. The reaction is likely the sum of 2 partial reactions, a ping pong kinetic mechanism; the reversibility of the first has been shown. Using HlyA labeled with selected radioactive fatty acyl groups, the reversibility of the second partial reaction will be shown. 3. Site-directed mutations of subcloned proHlyA, proHlyA- fragments containing either both or one or the other acylation sites, HlyA, and the respective acylated HlyA fragments will be studied to learn what defines an internal protein acylation site and what changes a protein undergoes upon acylation. Alterations in biological activity and fluorescence characteristics of site-directed fluoroprobes will be observed. E. coli hemolysin typifies one of a family of homologous, similarly activated, infectious protein toxins produced by diverse Gram negative bacteria. The toxins have remarkable organism and cellular specificities. The hemolysin scheme is an important recurring biological motif of rendering a protein toxic and secreting the infectious cellular-specific protein. Insight into the mechanism of this unique acyltransfer is of practical importance in providing a rationale for design of inhibitors of toxin activation as a potential therapeutic approach to severe infections with pathogenic E. coli where antibiotic therapy and subsequent toxin release worsen the clinical outcome.